Polysaccharide particle mixture

ABSTRACT

Tissue and other body structures may be protected using a dry, free-flowing, sterilized mixture of chitosan particles and oxidized polysaccharide particles in sealed packaging. The mixture may assist in returning an injured, inflamed or surgically repaired surface to a normal state, e.g., through one or more healing mechanisms such as modulation of an inflammatory response, phagocytosis, mucosal remodeling, reciliation or other full or partial restoration of normal function.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 14/324,889filed Jul. 7, 2014, now allowed, which is a continuation of applicationSer. No. 12/429,127 filed Apr. 23, 2009, now U.S. Pat. No. 8,802,652 B2,and which claims priority from U.S. Provisional Application Ser. No.61/047,580 filed Apr. 24, 2008, the disclosures of each of which areincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to polysaccharides and to materials for use inthe body.

BACKGROUND

Certain polysaccharide materials have been used for surgical repair ordrug delivery. Documents relating to such materials include U.S. Pat.No. 5,820,608 (Luzio et al.), U.S. Pat. No. 5,993,846 (Friedman et al.),U.S. Pat. No. 6,123,965 (Jacob et al.), U.S. Pat. No. 6,342,251 B1(lilum et al.), U.S. Pat. No. 6,706,690 B2 (Reich et al.), U.S. Pat. No.6,835,389 B1 (Dohi et al.) and U.S. Pat. No. 7,195,675 B2 (Okazaki etal.); U.S. Patent Application Publication No. US 2005/0208122 A1 (Allenet al.); Published PCT Application No. WO 93/21906 A (Brown UniversityResearch Foundation) and Weng et al., Rheological Characterization of inSitu Crosslinkable Hydrogels Formulated from Oxidized Dextran andN-Carboxyelhyl Chilosan, Biomacromolecules, 8, 1109-1115 (2007).Polysaccharide gels may be used as tissue sealants in ear, nose andthroat (ENT) procedures.

SUMMARY OF THE INVENTION

In order to avoid undue degradation during storage, it is desirable topackage polysaccharide gel materials in dry form (e.g., as a powder orsponge) and rehydrate the material just prior to use. Rehydrationsometimes presents difficulties. Some rehydrated materials provide gelsor sponges with poor physical properties. The physical properties of arehydrated gel may in some instances be improved via in situcrosslinking, but there may be an increased risk that an overlycrosslinked gel will inadvertently be dislocated (e.g., aspirated) intothe lungs or elsewhere in the form of large solid chunks. Some externalcrosslinking agents may damage tissue, or may cause residence timeswhich are excessively long or difficult to control.

The present invention provides, in one aspect, a composition comprisingfree-flowing rehydratable particles of substantially collagen-freedehydrothermally crosslinked polysaccharide. In one exemplary embodimentthe particles comprise a cellulose such as carboxymethylcellulose (CMC),and may provide a rehydrated gel having one or more desirable propertiesincluding rapid, clump-free rehydration; thixotropic behavior whensprayed or injected; high viscosity and cohesive gel character once inplace; controllable biodegradation properties, resistance to prematurebiodegradation and an ability to break down or be dislocated withoutproducing large solid chunks. In another exemplary embodiment theparticles comprise a blend of CMC and chitosan, and may provide arehydrated gel having one or more desirable properties like thoseprovided by the disclosed CMC rehydrated gels, together with one or moreadditional desirable properties including inherent antimicrobial (e.g.,bactericidal) behavior; hemostatic ability or the promotion of woundhealing. The disclosed rehydrated gels may assist in returning aninjured, inflamed or surgically repaired surface (e.g., a mucosal tissuesurface) to a normal state, e.g., through one or more healing mechanismssuch as modulation of an inflammatory response, phagocytosis, mucosalremodeling, reciliation or other full or partial restoration of normalfunction.

The invention provides in another aspect an implantable articlecomprising a rehydratable porous sponge comprising substantiallycollagen-free dehydrothermally crosslinked polysaccharide. The spongemay be packaged and sold in compressed form, may be trimmed to a desiredsize or shape for implantation at a treatment site, and may berehydrated prior to or following implantation. Exemplary embodiments ofthe disclosed sponge include sponges made from CMC and sponges made fromblends of CMC and chitosan.

The invention provides in another aspect a method for making apolysaccharide gel-forming composition, which method comprises providinga substantially collagen-free polysaccharide solution, drying thesolution to form a powder, and dehydrothermally crosslinking the powderto form free-flowing particles that will provide a polysaccharide gelwhen rehydrated. Exemplary embodiments of the disclosed method includemethods which make powders from CMC and from blends of CMC and chitosan.

The invention provides in another aspect a method for making animplantable article, which method comprises providing a substantiallycollagen-free polysaccharide solution, lyophilizing the solution to forma dried porous sponge, dehydrothermally crosslinking the sponge, andoptionally compressing the sponge, thereby forming an implantablearticle which will form a polysaccharide sponge when rehydrated.Exemplary embodiments of this disclosed method include methods whichmake sponges from CMC and from blends of CMC and chitosan.

The invention provides in another aspect a method for treating tissueand other body structures, which method comprises applying thereto a gelor sponge comprising rehydrated substantially collagen-freedehydrothermally crosslinked polysaccharide.

Rehydration may present additional difficulties. Some dry powdermaterials are prone to clumping when combined with water. The clumps canbe difficult to disperse and may plug syringes, cannula or spraynozzles. The invention provides, in yet another aspect, a method forconverting a dry powdered composition to a gel, which method comprisesdispersing free-flowing polysaccharide particles in a biocompatiblewater-miscible polar dispersant, and combining the resulting dispersionwith sufficient aqueous solvent for the particles to convert them to acohesive hydrogel. The polysaccharide particles may be crosslinked oruncrosslinked, and if crosslinked the crosslinking may be dehydrothermalcrosslinking or crosslinking carried out using a separate crosslinkingagent. The polysaccharide particles may be substantially collagen-free.The polysaccharide particles may be substantially a singlepolysaccharide or a blend of two or more polysaccharides. The cohesivehydrogel may be formed without visible clumps of unhydratedpolysaccharide. The disclosed method may be followed by a treatmentmethod including a step of injecting or spraying a layer of the cohesivehydrogel onto tissue (e.g., mucosal tissue) or other body structures.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic view showing the disclosed treatment method;

FIG. 2 is a perspective view of a dispensing instrument which may beused in the disclosed treatment method;

FIG. 3 is a perspective view of the disclosed sponge;

FIG. 4 is a perspective view of the disclosed sponge in a compressedstate;

FIG. 5 is a graph showing acid titration of three chitosan suspensions.

FIG. 6 and FIG. 7 are plots showing adhesion results for chitosan gelsdehydrothermally crosslinked at 90° C. and 120° C.

Like reference symbols in the various figures of the drawing indicatelike elements. The elements in the drawing are not to scale.

DETAILED DESCRIPTION

The following detailed description describes certain embodiments and isnot to be taken in a limiting sense. All weights, amounts and ratiosherein are by weight, unless otherwise specifically noted. The termsshown below have the following meanings:

The term “adhesion” refers to the sticking together of a body structureor prosthetic material to tissue, to the sticking together of tissue totissue with which it is in intimate contact for extended periods, or tothe formation of tissue that connects body structures, prostheticmaterials or tissues to one another across a normally open space.

The term “antimicrobial” refers to an ability to cause greater than a90% numeric reduction (viz., at least a 1-log order reduction) in apopulation of one or more of Staphylococcus aureus, Pseudomonasaeruginosa, Streplococcus pneumonia, Haemophilus influenzae or Moraxellacatarrhalis.

The terms “attached” and “adhered” when used in reference to a bacterialbiofilm and a surface mean that the biofilm is established on and atleast partially coats or covers the surface, and has some resistance toremoval from the surface. As the nature of this relationship is complexand poorly understood, no particular mechanism of attachment oradherence is intended by such usage.

The term “bacterial biofilm” means a community of bacteria attached to asurface, with the organisms in the community being contained within anextracellular polysaccharide (EPS) matrix produced by the bacteria.

The term “biocompatible” when used in reference to a substance meansthat the substance presents no significant deleterious or untowardeffects upon the body.

The term “biodegradable” when used in reference to a substance meansthat the substance will degrade or erode in vivo to form smallerchemical or physical species. Such degradation process may be enzymatic,chemical or physical.

The term “bioresorbable” when used in reference to a substance meansthat the substance is capable of being absorbed by the body.

The term “cohesive” when used in reference to a liquid or gel means thatthe liquid or gel when placed on a level surface will tend to (but neednot in all cases) stick to itself and form a unitary mass.

The term “comminuted” when used in reference to a particulate materialmeans that the particles have been fractured and reduced in size bycutting, grinding, pulverizing, triturating or other particle fracturingprocess employing externally-applied force.

The term “conformal” when used in reference to a composition applied totissue or other body structure means that the composition can form asubstantially continuous layer over an area to which the composition hasbeen applied.

The terms “detaching”, “removing” and “disrupting” when used inreference to a bacterial biofilm attached or adhered to a surface meanthat at least a significant amount of the biofilm initially present onthe surface no longer is attached or adhered to the surface. Noparticular mechanism of detachment, removal or disruption is intended bysuch usage.

The term “fluid” when used in reference to a substance means that thesubstance is a liquid having a loss modulus (G″) greater than itsstorage modulus (G′) and a loss tangent (tan δ) greater than 1.

The term “gel” when used in reference to a substance means that thesubstance is deformable (viz., is not a solid), G″ is less than G′ andtan δ is less than 1.

The term “gelation” when used with respect to formation of a gel layermeans the time at which G″ equals G′ and tan δ equals 1.

The term “hemostat” means a device or material which stops blood flow.

The term “hydrogel” when used in reference to a gel means that the gelis hydrophilic and contains water.

The term “hydrated” when used in reference to a device or substancemeans that the device or substance contains uniformly distributedchemically-bound water. A “fully hydrated” device or substance isincapable of taking up additional water of hydration. A “partiallyhydrated” device or substance is capable of taking up additional waterof hydration.

The term “inner ear” means the semicircular canals and cochlea.

The term “middle ear” means the region defined by the tympanic membrane,interior structures such as the ossicular chain, the surrounding liningand bordering structures such as the mastoid.

The term “mucoadhesive” when used in reference to a device or substancemeans that the device or substance will adhere to the mucus coveringepithelia.

The term “nasal or sinus cavities” refers to the various tissuesdefining the normally air-filled passages and chambers within the noseand sinus including but not limited to the nostrils or nares, the nasalconcha or turbinates, the frontal, ethmoid, sphenoid and maxillarysinuses, the sinus ostia and the nasopharnyx.

The term “polysaccharide” includes derivatives of polysaccharides andmodified polysaccharides, as well as derivatives of individualpolysaccharide species and modified individual polysaccharide species.For example, the term “carboxymethylcellulose” includescarboxymethylcellulose derivatives and modified carboxymethylcelluloses,the term “chitosan” includes chitosan derivatives and modifiedchitosans, and the term “starch” includes starch derivatives andmodified starches.

The term “protective” when used in reference to a layer of a compositionatop tissue or other body structure means that the layer may assist inreturning an injured, inflamed or surgically repaired tissue surface toa normal state, e.g., through one or more healing mechanisms such asmodulation of an inflammatory response, phagocytosis, mucosalremodeling, reciliation or other full or partial restoration of normalfunction.

The term “residence time” when used in reference to a protective gellayer atop tissue or other body structure means the time period duringwhich the gel layer or portion thereof remains in place in vivo undergross observation.

The term “solvating” means to form a solution or dispersion containing asolvent or other carrier within which a solute is dissolved orsuspended.

The term “substantially collagen-free” means containing a sufficientlylow amount of collagen so as not to pose a potential risk oftransmission of or infection with bovine spongiform encephalopathy (BSE)or variant Creutzfeldt-Jakob disease (vCJD).

The term “thin” when used in reference to a protective layer atop tissueor other body structure means having an average thickness less thanabout two millimeters.

Referring to FIG. 1, the disclosed treatment method may be performed forexample in the nasal or sinus cavities 100 of a patient, including themaxillary sinuses 110 a, 110 b and frontal sinuses 112 a, 112 b, whichmay be accessed through nares 114 a, 114 b. It should be noted thatexternal features of the patient, including nares 114 s, 114 b, areshown in dashed lines. When the patient suffers for example from chronicrhinosinusitis, one or more treatment sites such as treatment site 116associated with a surface of maxillary sinus 1100 may be medically or ifneed be surgically addressed. Treatment site 116 includes ciliatedepithelium of maxillary sinus 110 a and may include an associated layerof bacteria inhabiting an associated biofilm (not shown in FIG. 1). Thetreatment site need not be limited to natural tissue and may include anartificial structure (not shown in FIG. 1) such as a sinus packing orstent which may also be covered at least in part with a layer ofbacterial biofilm. If present, the biofilm may be removed using asolvating system (for example, the solvating system described in U.S.Patent Application Publication No. US 2007/0264310 A1) which may beapplied to treatment site 116 using an introducer 120 with anarticulatable delivery tube 122 containing an irrigation duct (hidden inFIG. 1) through which the solvating system may flow to a nozzle 124 atthe distal end of introducer 122 and thence to the treatment site. Thesolvating system and residues of the biofilm may be removed from thetreatment site via an aspiration duct (hidden in FIG. 1). The disclosedrehydrated gel composition may likewise be applied at the treatment siteusing the same or a different irrigation duct in introducer 120. Thoseskilled in the art will appreciate that the rehydrated gel (and if used,the solvating system) may be applied to the treatment site using othermethods or devices. Exemplary other methods include power spray or otherspray application, lavage, misting, mopping, wicking, dripping andtrephination and exemplary other devices include spray nozzles (e.g.,single component or multiple component spraying nozzles) and syringes(e.g., single barrel or multiple barrel glass or plastic syringes andbulb syringes). The treatment method may also be performed in otherparts of the body. The treatment method has particular utility innon-vascular applications, including treatment of tissues (e.g., mucosaltissues) or structures in or near the ears, nose or throat and openings,recesses, passageways or joints in the limbs or spinal column.

FIG. 2 shows an exemplary instrument 200 which may be used in thedisclosed treatment method. Instrument 200 includes a handle 202 and anintroducer 222 whose distal end 224 (referenced generally) includes aspray nozzle, irrigation and aspiration ducts (not separately numberedin FIG. 2). Instrument 200 can optionally further include a firstactuator assembly 226 (referenced generally) and a second actuatorassembly 228 (referenced generally). A control wheel 230 in firstactuator assembly 226 may be operable by a user to effectuate bending ofthe introducer 222, and a control wheel 232 in second actuator assembly228 may be operable by a user to effectuate movement or rotationrelative to introducer 222 of liquid sprayed from distal end 224 ofintroducer 222. Handle 202 serves generally as a housing for variousother components of instrument 200 and as a support for introducer 222.Handle 202 may have a pistol grip-like shape, defining a grip portion234 and a nose 236. Grip portion 234 is sized and shaped for grasping bya user's hand, whereas nose 236 is adapted for connection to introducer222. Trigger 238 and an associated sensor and valve (not shown in FIG.2) may be used to control the flow of the disclosed rehydrated gel (andif used, the disclosed solvating system) through irrigation tubing 240and thence through the spray nozzle in distal end 224 and onto thedesired treatment site. Trigger 238 may be provided with amultidirectional range of motion and associated with one or moreadditional sensors and valves (not shown in FIG. 2) to control removalfrom a treatment site of the solvating system, biofilm residue and otherdebris through the aspiration duct in distal end 224 and thence toaspiration tubing 242. Trigger 238 may also be used to control the flowof the disclosed rehydrated gel through a separate lumen in irrigationtubing 240 and thence through the spray nozzle in distal end 224 andonto the desired treatment site.

The applied rehydrated gel may fill the treatment site (e.g., a nasal orsinus cavity, or an opening, recess, passageway or joint in a portion ofthe limbs or spinal column), in which case the disclosed gel layer maybe very thick and not exposed to air or other nearby gases, and withdiffering thicknesses throughout the layer. The disclosed rehydrated gelmay also be applied as a thin film or other conformal coating in whichcase the disclosed gel layer may be relatively thin and exposed to airor other nearby gases, and with a substantially uniform thicknessthroughout the layer. The rehydrated gel composition provides aprotective layer which may be viscous, elastic or viscoelastic. Theprotective layer desirably adheres to mucosal or other natural tissues(e.g., cartilage or bone) at the treatment site and resists detachmentor other disruption until natural degradation or resorption of the gellayer takes place, e.g., after a residence time in vivo of from one dayto a few (e.g., 2, 3 or 4) days, weeks or months. Meanwhile bacterialrecolonization or reinfection may be significantly reduced or prevented,and improved healing and reciliation may take place. The protective gellayer may provide various therapeutic advantages including but notlimited to bacterial adhesion repellence, anti-infective properties,local immune modulation, tissue protection, reduction or elimination ofpain or bleeding, reduction in inflammation, optimization of environmentfor ciliary regrowth, reduction in adhesions to critical anatomy, andthe like. These advantages may arise due to a variety of mechanismsincluding a) killing bacteria, b) inhibiting bacterial colonization, c)inhibiting the adherence of bacteria to tissue, d) reducing tissuemorbidity or abscess formation, e) reducing or preventing diseaserecurrence (for example, specifically reducing the chronic inflammationrelated to bacterial toxin and EPS), f) coating and protecting tissueduring healing, such as by maintenance of a moist wound which promotesplatelet aggregation, or by closure of a dry wound without excessivescabrous formation, g) hemostasis, h) optimizing the environment forreciliation of the mucosa, i) speeding the growth or regrowth of ciliaand j) delivering therapeutic agent(s) to the treatment site. Desirablythe protective gel layer will adhere to a portion of the mucosa whileleaving the cilia in unadhered portions free to undergo natural rhythmiccilia motion (viz., cilia beating), will if desired also deliverantimicrobial agents or additional therapeutic agents, and desirablywill discourage or prevent bacteria from adhering to the treatment site.

FIG. 3 shows an example 30 of the disclosed sponge in an uncompressedstate, and FIG. 4 shows an example 40 of the disclosed sponge in acompressed state. In its uncompressed form prior to rehydration, sponge30 provides an essentially anhydrous porous polysaccharide matrix. Acompressed sponge such as sponge 40 may be formed before or afterdehydrothermal crosslinking, using a variety of techniques including apress with opposing platens, calendaring rollers, a plastic bagsubjected to external air pressure or internal vacuum, and othercompression techniques that may be envisioned by persons having ordinaryskill in the art. Either the compressed or uncompressed forms of thedisclosed sponge may be employed in medical procedures. Before placing asponge such as sponge 30 or sponge 40 in a treatment site, the spongemay be trimmed to a desired size or shape (using, for example, asuitable punch and die if trimming is done at a manufacturing site, orscissors or a scalpel if trimming is done at the time of placement). Theuntrimmed or trimmed sponge may then be rehydrated. If previouslycompressed, the sponge may be allowed to expand before, during or afterinsertion into the treatment site. The emplaced sponge may providevarious therapeutic advantages like those described above in connectionwith the protective gel layer.

A wide variety of polysaccharides may be employed in the disclosedrehydratable gel composition and in the disclosed sponge. In addition tocelluloses and chitosans, exemplary polysaccharides include agars,alginates, carrageenans, chitins, chondroitin sulfates, dextrans,galactomannans, glycogens, hyaluronic acids, starches and otherbiocompatible polysaccharides capable of being formed into a hydrogel orself-supporting sponge. Derivatives (including oxidized polysaccharidesand salts) and mixtures of polysaccharides (including derivatives) mayalso be used. Compositions containing mixtures of polysaccharides areespecially desirable in order to form hydrogels and sponges whoseproperties would not be provided using a single polysaccharide. Forexample compositions containing CMC and chitosan provide an especiallydesirable set of properties. Other desirable compositions include thosecontaining chitosan together with an alginate, hyaluronic acid orchondroitin sulfate. The chosen polysaccharide(s) desirably can becrosslinked via a dehydrothermal condensation reaction as described inmore detail below, and one or all of the polysaccharides in a mixture ofpolysaccharides may be so crosslinked. The chosen polysaccharide(s) alsodesirably are water soluble or may be rendered so, e.g., by suitableacidification.

A variety of celluloses may be employed in the disclosed rehydratablegel and in the disclosed sponge, including CMC, methylcellulose,ethylcellulose, hydroxypropyl cellulose, hydroxypropyl methyl celluloseand hemicellulose, as well as derivatives thereof including oxidizedcelluloses. Exemplary cellulosic materials may be obtained from avariety of commercial sources including Dow Wolff Cellulosics (forexample, the WALOCEL™ CRT line of sodium carboxymethylcelluloseproducts), Hercules, Inc. (for example, the AQUALON™ line of cellulosegum and carboxymethylcellulose products) and Sigma-Aldrich Co. (forexample, No. C4021 microgranular cellulose). The cellulosic materialdesirably is obtained in particulate form, for example, as free-flowinggranules whose average particle diameter is less than about 1 mm, lessthan about 100 μm, about 1 to about 80 μm, or less than 1 μm.

A variety of chitosans may be employed in the disclosed rehydratable geland in the disclosed sponge. Exemplary unmodified chitosans and theirsalts (including citrate, nitrate, lactate, phosphate, chloride andglutamate salts) may be obtained from a variety of commercial sourcesincluding KitoZyme S. A., Fluka Chemie AG, the NovaMatrix unit of FMCBioPolymer AS and Sigma-Aldrich Co. Chitosan may also be synthesized bydeacetylation of chitin (poly-N-acetyl-D-glucosamine) to eliminateacetyl groups on the nitrogen atom by hydrolysis. The resulting polymerhas a plurality of repeating units (e.g., about 30 to about 3000repeating units, about 60 to about 600 repeating units, or such otheramount as may be desired for the chosen end use) some or all of whichcontain deacetylated amino groups (e.g., about 30 to about 100% or about60 to about 95% of the total repeating units), with the remainingrepeating units (if any) containing acetylated amino groups. The polymeris cationic and may be regarded as being composed from glucosaminemonomers. The chitosan may have a variety of number average molecularweights, e.g., about 5 to about 2000 kDa, about 10 to about 500 kDa, orabout 10 to about 100 kDa. The chitosan may for example be an ultralowmolecular weight material having a number average molecular weight lessthan about 50 kDa, a low molecular weight material having a numberaverage molecular weight of about 50 to about 200 kDa, a mediummolecular weight material having a number average molecular weight ofabout 200 to about 500 kDa or a high molecular weight material having anumber average molecular weight greater than about 500 kDa. Chitosanderivatives may also be employed, for example derivatives in which oneor more hydroxyl or amino groups have been modified for the purpose ofaltering the solubility or mucoadhesion characteristics of thederivative. Exemplary derivatives include thiolated chitosans, andnon-thiolated chitosan derivatives such as acetylated, alkylated orsulfonated chitosans (for example O-alkyl ethers, O-acyl esters,cationized trimethyl chitosans and chitosans modified with polyethyleneglycol). Chitosan derivatives may be obtained from a variety of sources.For example, thiolated chitosans may be obtained from ThioMatrixForschungs Beratungs GmbH and Mucobiomer BiotechnologischeForschungs-und Entwicklungs GmbH or prepared by reaction of chitosanwith a suitable thiolated reactant, e.g., as described in Published PCTApplication No. WO 03/020771 A1 and in Roldo et al., Mucoadhesivethiolated chilosans as platforms for oral controlled drug delivery:synthesis and in vitro evaluation, European Journal of Pharmaceutics andBiopharmaceutics, 57, 115-121 (2004), Krauland et al., ViscoelasticProperties of a New in situ Gelling Thiolated Chitosan Conjugate, DrugDevelopment And Industrial Pharmacy, 31, 885-893 (2005),Bernkop-Schnürch, Thiomers: A new generation of mucoadhesive polymers,Advanced Drug Delivery Reviews, 57, 1569-1582 (2005) andBernkop-Schnürch et al., Thiomers: Preparation and in vitro evaluationof a mucoadhesive nanoparticulate drug delivery system, Internationaljournal of Pharmaceutics, 317, 76-81 (2006). The chitosan desirably isobtained in particulate form, for example, as free-flowing granuleswhose average particle diameter is less than about 1 mm, less than about100 μm, about 1 to about 80 m, or less than 1 μm.

Sources for and types of other polysaccharides (for example, agars,alginates, carrageenans, chitins, chondroitin sulfates, dextrans,galactomannans, glycogens, hyaluronic acids, starches) may be chosen bypersons skilled in the art based on selection characteristics similar tothose given above for celluloses and chitosans. When combined in amixture, the amounts of each polysaccharide may be varied widely toattain a desired combination of properties. For example, by altering theratio of two polysaccharides in a blend, the biodegradable orbioresorbable characteristics and residence time of the blend may bealtered. A mixture of two polysaccharides may for example contain about99 to about 1% of the first polysaccharide and about 1 to about 99% ofthe second polysaccharide, or about 80 to about 20% of the firstpolysaccharide and about 20 to about 80% of the second polysaccharide,or about 60 to about 40% of the first polysaccharide and about 40 toabout 60% of the second polysaccharide. Through appropriate selection ofthe types and amounts of polysaccharides in a mixture, rehydratable gelsand sponges with tunable properties may be obtained. For example, ablend of CMC and chitosan may have good bacteriostatic performance dueto the chitosan and controlled, sustained and tunable degradation ratesdue to the CMC, whereas chitosan used alone may form a gel or spongehaving inherently poor mechanical and resorbtive properties and CMC usedalone may form a gel or sponge lacking bactericidal properties.

The disclosed rehydratable gel composition and sponge are substantiallycollagen-free. Desirably the rehydratable gel composition and sponge aresufficiently free of collagen (e.g., containing no collagen at all) soas to be saleable worldwide for use without restriction in humans.

The disclosed rehydratable gel composition and sponge optionally arecrosslinked before being packaged and sent to end users. Crosslinkingpreferably is carried out using a dehydrothermal crosslinking process.For the disclosed rehydratable gel this preferably is done bydehydrothermally crosslinking a mass of free-flowing rehydratablepolysaccharide particles to form free-flowing rehydratable crosslinkedpolysaccharide particles. In other words, the particles preferably arethemselves individually crosslinked while still remaining free-flowingand capable of later rapid dissolution and rehydration. For thedisclosed sponge, crosslinking preferably is done by dehydrothermallycrosslinking a shaped porous article which has been made by placing asolution of the desired polysaccharide in a suitable mold andlyophilizing the solution to form a porous solid having a shapecorresponding to the desired uncompressed sponge shape. In other words,the sponge preferably is shaped and made porous prior to crosslinking.

Dehydrothermal crosslinking is in effect a solid state crosslinkingprocess in which a material is exposed to one or both of heat andreduced pressure to cause initial dehydration followed by loss ofadditional water and formation of crosslinking bonds via an inter- orintra-molecular condensation process. It is not necessary to addexternal cross-linking agents, and in the case of the disclosedparticles the presence of such agents could make it difficult to retaintheir free-flowing nature. Dehydrothermal crosslinking desirablyinvolves dehydrating the product to be crosslinked to a moisture contentless than about 1%, and using sufficient additional heat or vacuum toachieve a desired crosslink density. For example, in the absence ofvacuum, temperatures above about 80° C., above about 90° C., above about100° C. or above about 120° C. may be employed, with higher temperaturesgenerally providing faster reaction rates. The polysaccharide desirablyis not heated to an extent sufficient to cause browning, and accordinglytemperatures less than 160° C. or less than 150° C. are preferred.Fairly long heating times may be needed at ambient pressure, forexample, about 40 hours at 140-150° C. plus about total 20 hours forwarmup and cooldown. When reduced pressure is used, lower temperaturesmay be employed and a pressure of at most about 1 mm Hg, and preferablyat most about 10⁻³ mm Hg may be preferred. Thus the higher thetemperature, the lower the required vacuum or beating time required toarrive at a given crosslink density, and vice versa. It is accordinglydifficult to specify an exact heating time or range of heating times,although times of at least about 10 hours, at least about 20 hours, atleast about 30 hours or about 40 to about 60 hours (not counting thetimes required for warmup and cooldown) may be employed. In many casesit will suffice to determine the heating time, temperature and pressureempirically, for example by using the Gel Retention Time test shown inthe Examples to evaluate whether an appropriate degree of crosslinkinghas been obtained. For an uncrosslinked sample, the Gel Retention Timemay for example be 1 day or less, whereas by using an appropriate degreeof dehydrothermal crosslinking the Gel Retention Time may for example beextended to at least 2 days and preferably about 3 to about 7 days. Theuncrosslinked sample may also tend to rehydrate less rapidly, absorbmore water or form a more viscous gel or paste than its dehydrothermallycrosslinked counterpart. In comparison to a conventionally crosslinkedmaterial ground into powder form, dehydrothermally crosslinked particlesmay be non-comminuted, may be crosslinked due to a condensation reaction(e.g., a dehydration reaction leading to the loss of water, or areaction leading to the loss of another small molecule such as hydrogenchloride, methanol or acetic acid) rather than due to other crosslinkingreactions (e.g., reactions involving addition polymerization (e.g. ofvinyl groups), ionic reactions, or reactions involving sulfide or aminegroups). In comparison to a conventionally crosslinked material groundinto powder form, dehydrothermally crosslinked particles may also have anarrower polydispersity index, lower number average molecular weight,the capability to undergo further crosslinking, lower production costsand lower manufacturing capital requirements.

When two or more polysaccharides are employed to make the disclosedrehydratable gel, the dehydrothermal crosslinking process may beperformed on one or on more than one of the polysaccharides before theparticles have been blended. This permits customization of propertiessuch as gelation behavior, gelation time and degradation time followingimplantation, by varying properties including the crosslinking time,temperature or vacuum for each polysaccharide component followed byblending of the crosslinked (or if desired, uncrosslinked) componentsafter completion of the dehydrothermal crosslinking reaction(s) on theindividual blend components. If desired, the resulting blend may besubjected to an additional dehydrothermal crosslinking reaction. Theparticles could also be kept separate and later mixed by an end user,although this will normally be less convenient than forming the mixtureat a manufacturing site.

Dehydrothermal crosslinking conditions for the disclosed sponges aresimilar to those which may be employed for the polysaccharide particles.When two or more polysaccharides are employed to make the disclosedsponge, a mixture of the polysaccharides may be formed in solution,lyophilized and dehydrothermally crosslinked. As another approach, oneor more of the polysaccharides in such a mixture may be dehydrothermallycrosslinked, and the remaining polysaccharide(s) in such mixture may beimbibed into the dehydrothermally crosslinked polymer and the resultingswelled article may be lyophilized to form a sponge. These and otherrelated approaches can permit differing degrees of propertycustomization.

The disclosed rehydratable gel composition and sponge typically will besubjected to sterilization and placed in suitable sealed packaging (forexample, a syringe, vial or bag made of a suitable material) prior toshipment to an end user. Additional property customization may becarried out by using a sterilization procedure such as gamma radiationor electron beam (E-Beam) processing to cause controlled chain scission.Cold ionizing radiation sterilization (e.g., cold E-Beam sterilization)may be employed to limit the degree of chain scission, as discussed inInternational Application No. PCT/US2009/041593 (corresponding toInternational Publication No. WO 2009/132229 A2), filed even dateherewith.

The disclosed rehydratable gel composition and sponge may be rehydratedprior to placement or insertion in a treatment site, or may be placedwhile in a dry state and then rehydrated in silu (e.g., via the additionof an externally-supplied rehydrating fluid, by the uptake of endogenousfluids, or both). Rehydrating the sponge normally is relativelystraightforward, and may be carried out by immersing or saturating thesponge with water or an aqueous solution containing any other desiredingredients. For example, normal saline solution may be a preferred andreadily available rehydration solution, and other materials such asphosphate buffered saline (PBS) may be used if desired. Rehydrating therehydratable gel particles may as noted above present additionaldifficulties due to the tendency of some dry powdered materials to formclumps when combined with water. Clumping may however be avoided bydispersing the rehydratable gel particles in a biocompatiblewater-miscible polar dispersant, followed by mixing the dispersion withsufficient aqueous particle solvent (viz., a water-based solvent for theparticles) to convert the particles to a cohesive hydrogel. Thedispersant is a thus a sufficiently poor solvent for the particles sothat the mixture of particles and dispersant will not form a truesolution. The particles in such a dispersion desirably are sufficientlysmall so that the dispersion is stable or quasi-stable (e.g., acolloidal dispersion or a reasonably persistent suspension) after theparticles and dispersant have been agitated, e.g., by swirling themtogether. Without being bound by theory, the addition of the aqueousparticle solvent is believed to permit rehydration to occurapproximately simultaneously at the surface of each suspended particlevia dissolution of the surrounding dispersant into the aqueous particlesolvent phase, thereby permitting formation of a cohesive hydrogelwithout forming visible clumps of unhydrated polysaccharide. In thisfashion a dispersed polysaccharide may be combined with water or anaqueous solution to form a clump-free hydrogel even though the drypowdered polysaccharide would not ordinarily do so. In many instancesthe disclosed method may be used to prepare a satisfactory clump-freegel using passage between two syringes, mild agitation or other simplemixing techniques without requiring the use of a mechanical stirrer. Thedisclosed mixing method may also permit formation of very concentratedhydrogels which could not be obtained by merely mixing a powderedpolysaccharide with water or acidified water. The polysaccharideconcentration typically will depend on the chosen molecular weight, andmay for example be about 1 to about 20%, about 1 to about 10% or about 1to about 5% of the rehydrated gel. The gel may desirably form in lessthan 30 minutes, less than 20 minutes, less than 10 minutes, less than 5minutes, less than 1 minute or even essentially immediately afterrehydration. For polysaccharides which do not immediately rehydrate, itmay be desirable to saturate the powder and inject it before thepolysaccharide has become too viscous to spray or otherwise dispensethrough a small orifice.

The selection of dispersant and aqueous particle solvent may depend uponthe chosen polysaccharide. For polysaccharides such as chitosan whichhave relatively poor solubility in pure water but which become solublewhen the water is acidified, deionized water may be used as thedispersant and acidified water may be used as the aqueous particlesolvent. Other combinations of dispersant and aqueous solvent may alsobe used. For example, ethanol, isopropanol or acetone may be used as thedispersant for many polysaccharides (including chitosan and blendscontaining chitosan) and deionized water, normal saline solution or PBSmay be used as the aqueous particle solvent.

The disclosed rehydratable gel particles may as noted above becrosslinked or uncrosslinked, and if crosslinked the crosslinking may bedehydrothermal crosslinking or crosslinking carried out using a separatecrosslinking agent (for example, genipin, oxidized polysaccharide orglutaraldehyde). When crosslinked using a separate crosslinking agent,the resulting polymer may optionally be lyophilized and if need becomminuted to provide free-flowing particles.

The disclosed rehydratable gel composition and sponge may optionallyinclude a variety of other ingredients before or after rehydration.Exemplary other ingredients include other solvents, acids, bases,buffering agents, antimicrobial agents, therapeutic agents and otheradjuvants. An acid, base or buffering agent may for example maintain thegel at an appropriate pH for contacting human tissue, e.g., a pH greaterthan 5, a near-neutral pH, or a pH less than 8.5. Exemplary bufferingagents include barbitone sodium, glycinamide, glycine, potassiumchloride, potassium phosphate, potassium hydrogen phthalate, sodiumacetate, sodium citrate, sodium phosphate and their conjugate acids.

The disclosed rehydratable gel composition and sponge desirably areinherently antimicrobial without requiring addition of a separateantimicrobial agent. A separate antimicrobial agent may be employed ifdesired. A useful list of such antimicrobial agents may be found, forexample, in the above-mentioned U.S. Patent Application Publication No.US 2007/0264310 A1.

Exemplary therapeutic agents which may be employed in the disclosedrehydratable gel composition and sponge include any material suitablefor use at the intended treatment site including analgesics,anti-cholinergics, anti-fungal agents, antihistamines, steroidal ornon-steroidal anti-inflammatory agents, anti-parasitic agents, antiviralagents, biostatic compositions, chemotherapeutic/antineoplastic agents,cytokines, decongestants, hemostatic agents (e.g., thrombin),immunosuppressors, mucolytics, nucleic acids, peptides, proteins,steroids, vasoconstrictors, vitamins, mixtures thereof, and othertherapeutic materials that will be known to those skilled in the art. Auseful list of such therapeutic agents may be found, for example, in theabove-mentioned U.S. Patent Application Publication No. US 2007/0264310A1.

Other adjuvants that may be included in the disclosed rehydratable gelcomposition and sponge include dyes, pigments or other colorants (e.g.,FD & C Red No. 3, FD & C Red No. 20, FD & C Yellow No. 6, FD & C BlueNo. 2, D & C Green No. 5, D & C Orange No. 4, D & C Red No. 8, caramel,titanium dioxide, fruit or vegetable colorants such as beet powder orbeta-carotene, turmeric, paprika and other materials that will be knownto those skilled in the art); indicators; flavoring or sweetening agentsincluding but not limited to anise oil, cherry, cinnamon oil, citrus oil(e.g., lemon, lime or orange oil), cocoa, eucalyptus, herbal aromatics(e.g., clove oil, sage oil or cassia oil), lactose, maltose, menthol,peppermint oil, saccharine, sodium cyclamate, spearmint oil, sorbitol,sucrose, vanillin, wintergreen oil, xylitol and mixtures thereof;antioxidants; antifoam agents; and theology modifiers includingthickeners and thixotropes. The disclosed rehydratable gel compositionand sponge desirably do not contain ingredients which might potentiallyharm patient tissues or structures, e.g., mucosal tissues in the nasalor sinus cavities.

In those instances where it is desirable to remove water from tissue,e.g., to remove fluid from polyps or edematous tissue, a hyperosmolaragent may be employed in the disclosed rehydratable gel composition andsponge. Exemplary hyperosmolar agents include furosemide, sodiumchloride gel and other salt preparations that draw water from tissue orsubstances which directly or indirectly change the osmolar content ofthe mucous layer. Where sustained release or delayed release of atherapeutic agent is desirable, a release agent modifier may also beincluded.

The disclosed rehydratable gel composition and sponge may desirably beused as a part of a multi-step treatment regimen which disrupts abacterial biofilm and discourages its return. For example, a series ofsteps that may be broadly classified as Cleansing/Disrupting, Killing,Aerating, Protecting/Coating, and Healing may be carried out. TheCleansing/Disrupting step may be carried out by administering asolvating system as discussed above in connection with FIG. 1 and FIG.2. The Killing step may be carried out by applying a suitableantimicrobial agent to the treatment site. This may for example beaccomplished by including an antimicrobial agent in the solvatingsystem, as a separately-applied composition, or in both the solvatingsystem and in a separately-applied composition. An antimicrobial agentmay also be applied or administered post operatively. The Aerating stepmay be carried out by providing air passageways or improving airpassageways to the treated tissues by opening occluded or partiallyoccluded passages, e.g., the sinuses or sinus ostia for nasalapplications. This may for example be accomplished by surgicallyremoving obstructive tissue structures or by manually displacing suchstructures. The Protecting/Coating step may be carried out by coating atleast part of the thus-treated tissue with the disclosed gel compositionor by covering at least part of the thus-treated tissue with thedisclosed sponge. The Healing step may be carried out by allowing thecleansed, protected and sealed tissue surface to undergo a return to anormal state, e.g., through one or more healing mechanisms such asmodulation of an inflammatory response, phagocytosis, mucosalremodeling, reciliation or full or partial restoration of normalfunction. The multi-step treatment regimen may include or be followed bya Clearing step in which the gel composition or sponge is sufficientlybiodegradable or bioresorbable to disappear from the treatment site in adesired time period, e.g., more than 1 day, more than 3 days, or about 4to 7 days, and desirably without shedding large solid chunks. Thedisclosed method may advantageously be accomplished without requiringsurgery, for example by applying and removing the optional solvatingsystem through normal aspiration/suction techniques or by simpleflushing of affected tissue followed by application of the disclosed gelcomposition or sponge. A comparable series of steps may be performed ina multi-step treatment regimen in a portion of the middle or inner ear.Further details regarding such a regimen may be found in U.S. PatentApplication Publication No. US 2007/0264310 A1.

The invention is further illustrated in the following non-limitingexamples.

Example 1 Dehydrothermally Crosslinked CMC

Powdered sodium carboxymethylcellulose (CMC 52MSC from EmergingTechnologies, Inc.) was combined with 1% titanium dioxide anddehydrothermally crosslinked by warming to 140° C. over 10 hours,heating at 150-160° C. for 40 hours, and cooling to ambient temperatureover 10 hours. A free-flowing, white powder with an average particlediameter of about 40 μm was obtained. The powder was packaged in 1.5gram portions in polyurethane pouches and E-Beam sterilized at 30 kGy.

Example 2 Syringe Rehydration and Dispensing

A 0.7 g portion of the Example 1 CMC powder was combined with 3 mLdeionized water in a 10 mL LUER-LOK™ syringe (from Becton, Dickinson andCo.). The syringe was connected to a second such 10 mL syringe using aLUER™ connector (from Becton, Dickinson and Co.) and the syringeplungers were alternately depressed in an effort to rehydrate thepowder. A poorly-mixed mass containing many clumps was obtained, and theclumps occluded the passage between the syringes. In a second run, a 0.7g portion of the Example 1 CMC powder was combined with 0.5 mL ethanoland shaken, resulting in an apparently stable suspension. A 3 mL portionof deionized water in the second syringe was added to the suspension andthen the syringe plungers were alternately depressed to mix theingredients, resulting in the gradual formation of a clear, homogenoushydrogel which was completely free of visible unhydrated CMC particles.The hydrogel could readily be injected through a 2.7 mm inside diameter,10 cm long cannula onto a target surface, resulting in a thick, highlyviscous uniform gel with no noticeable clumps. The gel looked somewhatlike an amorphously-shaped sponge.

Example 3 Adhesion

Samples of the Example 2 gel were placed between collagen-coated pins inan MTS tensile testing machine (MTS Systems Corp.), and evaluated at aseparation rate of 25.4 mm/min to determine tensile adhesive strengthfor the gel-collagen bond. A value of about 55 kPa was obtained, whereasonly about 35 kPa was obtained using a gel made with uncrosslinked CMCpowder.

Example 4 Gel Retention Time

A 1.5 g portion of the Example 1 powder was rehydrated to form aclump-free gel using the method of Example 2. The gel was submerged in200 mL PBS, then poured onto a 150 μm sieve, allowed to drain andweighed. The gel was returned to the collected PBS solution, storedovernight and the sieve and reweighing procedure repeated for a total ofthree times at which point the gel had disappeared. The resulting 3 daytest duration was recorded as the Gel Retention Time. A gel made fromthe Example 1 CMC powder without dehydrothermal crosslinking had a GelRetention Time of less than 1 day.

Example 5 Chitosan Suspensions and Gels

High molecular weight powdered chitosan obtained from crab shells(BIOCHEMICA™ high viscosity chitosan from Sigma-Aldrich Co.) wassuspended in deionized water at addition levels of 10, 25 and 50 g/L. Astirred 50 mL portion of each suspension was titrated with 1M HCl. Asshown in FIG. 5, where the 10, 25 and 50 g/L suspensions arerespectively identified as curves A, B and C, the pH value decreasedgradually with the addition of acid. The pH value passed through aninflection point at about pH 6 and less than 1 mL of acid addition,corresponding to the start of noticeable gel formation. High viscositygels were obtained by the time the pH value passed through anotherinflection point, at approximately pH 4.5 and at about 2, 3 or 5 mL ofacid addition for curves A, B and C, respectively. At yet lower pHvalues the chitosan had by then become fully hydrated and the additionalacid further acidified the water and diluted the gel.

In a separate run, a 1% solution of the high molecular weight chitosanwas formed by adding the chitosan powder to deionized water containing1% acetic acid. The solution had a 453 cP viscosity when evaluated at37° C. using a BROOKFIELD™ DV-II+ Pro cone and plate viscometer equippedwith a CP-52 cone spindle. A similarly-prepared 1% solution of lowmolecular weight chitosan (BIOCHEMICA low viscosity chitosan fromSigma-Aldrich Co.) had a 185 cP viscosity.

Example 6 Dehydrothermally Crosslinked Chitosan Adhesion

Using the method of Example 1, low and high molecular weight powderedchitosan was dehydrothermally crosslinked at 90° C. for 10 hours or at120° C. for 20 hours, added to varying amounts of 1M HCl to form gelsand evaluated for adhesion using the method of Example 3. FIG. 6 showsthe adhesion results for chitosan crosslinked at 90° C. and FIG. 7 showsthe adhesion results for chitosan crosslinked at 120° C.

In a separate run, medium molecular weight chitosan was dehydrothermallycrosslinked at 120° C. for 40 hours using the method of Example 1 andevaluated for adhesion using the method of Example 2. A value of about65 kPa was obtained, whereas only about 41 kPa was obtained using a gelmade with uncrosslinked CMC powder.

Example 7 Polysaccharide Blends

In a series of runs, powdered chitosan (ultralow molecular weightchitosan oligosaccharide lactate, or BIOCHEMICA™ low or medium viscositycrab shell chitosan, all from Sigma-Aldrich Co.) was employed by itself,in a blend of chitosans, or in a blend with the Example 1 powdered CMC,and mixed with water acidified with varying amounts of HCl or aceticacid. The resulting mixtures were stirred for five minutes using apowered stirrer. Doing so enabled the antimicrobial testing describedbelow, but would be much less convenient if required to be performedprior to surgery. The stirred solutions were evaluated to determine theantimicrobial activity of the resulting gels versus E. Coli, S. Aureus,P. Aeruginosa and in some instances S. Pneumoniae. The antimicrobialactivity evaluation used a modified zone of inhibition plate procedure,in which a small quantity of the gel was streaked across agar plateswhich had been freshly streaked with the desired microbe. Unlike atraditional zone of inhibition test, in which an antibiotic-soaked paperdisk is placed against the agar surface and the antibiotic diffusesoutwardly through the agar, the viscous nature of the gel discouragedsuch diffusion. The results were thus somewhat Boolean in nature, andmainly provided an indication as to whether or not inhibition hadoccurred. The results are shown below in Table 1.

TABLE 1 Antimicrobial Activity Evaluation Chitosan Run (amount and CMCMicrobial Inhibition No. type) (amount) Acidification¹ E. Coli S. AureusP. Aeruginosa S. Pneumoniae  1  100% LMW²    pH 4.5 Yes Yes Yes  2 100%LMW  pH 6 No Mild No  3  100% MMW³   pH 4.5 No No No  4 100% MMW pH 6 NoMild No  5 20% LMW 80%   pH 4.5 Yes Yes Yes   6⁴ 20% LMW 80%   pH 4.5Yes Yes Yes  7 40% LMW 60%   pH 4.5 Yes Yes Yes  8 60% LMW 40%   pH 4.5Yes Yes Yes  9 80% LMW 20%   pH 4.5 Yes Yes Yes  10⁴ 80% LMW 20%   pH4.5 Yes Yes Yes 11 20% LMW 80% pH 5 Yes Yes Yes 12 40% LMW 60% pH 5 YesYes Yes 13 60% LMW 40% pH 5 Yes Yes Yes 14 80% LMW 20% pH 5 Yes Yes Yes15 20% LMW 80%   pH 5.5 Yes Yes Yes 16 40% LMW 60%   pH 5.5 Yes Yes Yes17 60% LMW 40%   pH 5.5 Yes Yes Yes 18 80% LMW 20%   pH 5.5 Yes Yes Yes19 20% LMW 80%  0.1% acetic acid Yes Yes Yes Yes 20 20% LMW 80% 0.12%acetic acid Yes Yes Yes Yes 21 20% LMW 80% 0.14% acetic acid Yes Yes YesYes 22 20% LMW 80% 0.16% acetic acid Yes Yes Yes Yes 23 20% LMW 80%0.18% acetic acid Yes Yes Yes Yes 24 20% LMW 80%  0.2% acetic acid YesYes Yes Yes 25 20% LMW 80%  0.2% acetic acid Yes Yes Yes Yes 26 20% LMW80%  0.2% acetic acid No No No No 27 50% LMW 50%  0.2% acetic acid YesYes Yes Yes 28 20% LMW +  5%  0.2% acetic acid Yes Yes Yes Yes  75% MMW29 20% LMW + pH 5 Yes Yes Yes  80% MMW 30 10% LMW + pH 5 Yes Yes Yes 90% MMW 31  5% LMW + pH 5 Yes Yes Yes  95% MMW 32  3% LMW + pH 5 YesYes Yes  97% MMW 33  2% LMW + pH 5 Yes Yes Yes  98% MMW 34  1% LMW + pH5 Yes Yes Yes  99% MMW 35 0.5% LMW +  pH 5 Yes Yes Yes 99.5% MMW  36100% MMW pH 5 Yes Yes Yes 37 100% ULMW⁵ pH 5 Yes Yes Yes Yes ¹Theindicated pH values are the pH at the start of rehydration, as obtainedusing HCl. Where acidification was performed using acetic acid, theamount of added acid is shown. ²“LMW” is low molecular weight chitosan.³“MMW” is medium molecular weigh chitosan. ⁴Gel sample aged 48 hoursprior to microbial inhibition test. ⁵“ULMW” is ultralow molecular weighchitosan.

The results in Table 1 show that chitosan and blends of chitosan withthe polysaccharide carboxymethylcellulose may be rehydrated to providegel layers with inherent antimicrobial properties.

Example 8

A free-flowing crosslinked powder may be prepared by dissolving a drypowdered polysaccharide polymer such as carboxymethyl chitosan in waterto produce a viscous solution containing about 5 wt. % polymer. Acrosslinker solution containing 10 wt. % dialdehyde starch or 0.1 wt. %glutaraldehyde may be quickly mixed with the polymer solution by placingeach solution in a 10 mL LUER-LOK™ syringe (from Becton, Dickinson andCo.), connecting the syringes to one another using a LUER™ connector(from Becton, Dickinson and Co.) and alternately depressing the syringeplungers to exchange the fluids between the two syringes several times.After a short dwell period during which crosslinking takes place, acohesive gel should be obtained. The gel may be converted to particlesby freezing and lyophilize the frozen gel, followed by grinding thelyophilization product.

Example 9

A free-flowing crosslinked powder may also be prepared by soaking a drypowdered polysaccharide polymer in a nonsolvating liquid crosslinkingagent or nonsolvating crosslinker solution. The dry powderedcarboxymethyl chitosan starting material used in Example 8 may be soakedin ethylene glycol diglycidyl ether (e.g., E27203 ethylene glycoldiglycidyl ether from Sigma-Aldrich) for sufficient time to permitcrosslinking to occur. The resulting mass of free-flowing, crosslinkedparticles may be washed with methanol to remove residual crosslinkingagent and dried using gentle heat. Depending on the chosenpolysaccharide, a variety of crosslinkers may be employed. For example,ethylene glycol diglycidyl ether may be replaced with hexamethylenediglycidyl ether or other glycidyl crosslinker reactive towards hydroxylor amine groups. If the polysaccharide contains primary amine groups,appropriately reactive crosslinkers such as dialdehyde starch, oxidizedmethyl cellulose or glutaraldehyde may be employed.

Example 10

Equal volumes of a 2.5 wt. % solution of carboxymethyl chitosan in PBSand 10 wt. % dialdehyde starch were quickly mixed using two 3 mLsyringes like the 10 mL syringes described in Example 8. The resultingcohesive hydrogel was expelled from the back of the syringes to providea mass that maintains its shape.

Example 11

Equal volumes of a 5 wt. % solution of carboxymethyl chitosan in PBSwere crosslinked via E-beam radiation both when frozen and at roomtemperature. The resulting hydrogels were lyophilized into readilypowderable sponges. When placed in PBS, the sponges persisted for threeto seven days.

In addition to a composition comprising free-flowing rehydratableparticles of substantially collagen-free dehydrothermally crosslinkedpolysaccharide, other embodiments of the invention include likecompositions:

-   -   wherein the particles contain substantially a single        polysaccharide;    -   wherein the particles comprise a mixture of polysaccharides;    -   wherein the polysaccharide comprises cellulose, chitosan, agar,        alginate, carrageenan, chitin, chondroitin sulfate, dextran,        galactomannan, glycogen, hyaluronic acid, starch or mixture        thereof,    -   wherein the polysaccharide comprises an oxidized polysaccharide        or salt;    -   wherein the polysaccharide comprises carboxymethylcellulose;    -   wherein the polysaccharide is substantially only        carboxymethylcellulose;    -   wherein the polysaccharide comprises chitosan;    -   wherein such chitosan is unmodified;    -   wherein the polysaccharide comprises a mixture of chitosan and        another polysaccharide;    -   wherein the polysaccharide comprises a mixture of        carboxymethylcellulose and chitosan; or    -   further comprising a sealed syringe with which the particles can        be rehydrated and dispensed.

In addition to an implantable article comprising a rehydratable poroussponge comprising substantially collagen-free dehydrothermallycrosslinked polysaccharide; other embodiments of the invention includelike articles:

-   -   wherein the polysaccharide is substantially a single        polysaccharide;    -   wherein the polysaccharide comprises a mixture of        polysaccharides;    -   wherein the polysaccharide comprises cellulose, chitosan, agar,        alginate, carrageenan, chitin, chondroitin sulfate, dextran,        galactomannan, glycogen, hyaluronic acid, starch or mixture        thereof;    -   wherein the polysaccharide comprises an oxidized polysaccharide        or salt;    -   wherein the polysaccharide comprises carboxymethylcellulose;    -   wherein the polysaccharide is substantially only        carboxymethylcellulose;    -   wherein the polysaccharide comprises chitosan;    -   wherein such chitosan is unmodified;    -   wherein the polysaccharide comprises a mixture of chitosan and        another polysaccharide;    -   wherein the polysaccharide comprises a mixture of        carboxymethylcellulose and chitosan; or    -   wherein the sponge is compressed and may be trimmed to a desired        size or shape, rehydrated and allowed to expand after        rehydration.

In addition to a method for making a polysaccharide gel-formingcomposition, which method comprises:

-   -   a) providing a substantially collagen-free polysaccharide        solution;    -   b) drying the solution to form a powder, and    -   c) dehydrothermally crosslinking the powder to form free-flowing        particles that will provide a polysaccharide gel when        rehydrated,        other embodiments of the invention include like methods:    -   wherein the particles contain substantially a single        polysaccharide;    -   wherein the polysaccharide comprises a mixture of        polysaccharides;    -   wherein the polysaccharide comprises cellulose, chitosan, agar,        alginate, carrageenan, chitin, chondroitin sulfate, dextran,        galactomannan, glycogen, hyaluronic acid, starch or mixture        thereof;    -   wherein the polysaccharide comprises an oxidized polysaccharide        or salt;    -   wherein the polysaccharide comprises carboxymethylcellulose;    -   wherein the polysaccharide is substantially only        carboxymethylcellulose;    -   wherein the polysaccharide comprises chitosan;    -   wherein such chitosan is unmodified;    -   wherein the polysaccharide comprises a mixture of chitosan and        another polysaccharide;    -   wherein the polysaccharide comprises a mixture of        carboxymethylcellulose and chitosan; or    -   further comprising packaging the particles in a sealed syringe        with which the particles can be rehydrated and dispensed.

In addition to a method for making an implantable article, which methodcomprises:

-   -   a) providing a substantially collagen-free polysaccharide        solution,    -   b) lyophilizing the solution to form a dried porous sponge, and    -   c) dehydrothermally crosslinking the sponge,        thereby forming an implantable article which will form a        polysaccharide sponge when rehydrated,        other embodiments of the invention include like methods:    -   wherein the sponge contains substantially a single        polysaccharide;    -   wherein the sponge contains a mixture of polysaccharides;    -   wherein the polysaccharide comprises cellulose, chitosan, agar,        alginate, carrageenan, chitin, chondroitin sulfate, dextran,        galactomannan, glycogen, hyaluronic acid, starch or mixture        thereof;    -   wherein the polysaccharide comprises an oxidized polysaccharide        or salt;    -   wherein the polysaccharide comprises carboxymethylcellulose;    -   wherein the polysaccharide is substantially only        carboxymethylcellulose;    -   wherein the polysaccharide comprises chitosan;    -   wherein such chitosan is unmodified;    -   wherein the polysaccharide comprises a mixture of chitosan and        another polysaccharide;    -   wherein the polysaccharide comprises a mixture of        carboxymethylcellulose and chitosan;    -   further comprising compressing the dried porous sponge before or        after dehydrothermal crosslinking; or    -   further comprising packaging the implantable article in a sealed        container.

In addition to a method for treating mucosal tissue and other bodystructures, which method comprises applying thereto a gel or spongecomprising rehydrated substantially collagen-free dehydrothermallycrosslinked polysaccharide, other embodiments of the invention includelike methods:

-   -   comprising placing the gel or sponge in a nasal or sinus cavity;    -   comprising placing the gel or sponge in a middle or inner ear;    -   comprising placing the gel or sponge in an opening, recess,        passageway or joint in a limb;    -   comprising placing the gel or sponge in an opening, recess,        passageway or joint in a spinal column;    -   comprising spraying the gel;    -   further comprising trimming the sponge to a desired size or        shape, rehydrating the trimmed sponge and allowing it to expand;    -   wherein the wherein the polysaccharide is substantially a single        polysaccharide;    -   wherein the polysaccharide comprises a mixture of        polysaccharides;    -   wherein the polysaccharide comprises cellulose, chitosan, agar,        alginate, carrageenan, chitin, chondroitin sulfate, dextran,        galactomannan, glycogen, hyaluronic acid, starch or mixture        thereof,    -   wherein the polysaccharide comprises an oxidized polysaccharide        or salt;    -   wherein the polysaccharide comprises carboxymethylcellulose;    -   wherein the polysaccharide is substantially only        carboxymethylcellulose;    -   wherein the polysaccharide comprises chitosan;    -   wherein such chitosan is unmodified;    -   wherein the polysaccharide comprises a mixture of chitosan and        another polysaccharide; or    -   wherein the polysaccharide comprises a mixture of        carboxymethylcellulose and chitosan.

In addition to a method for converting a dry powdered composition to agel, which method comprises:

-   -   a) dispersing free-flowing polysaccharide particles in a        biocompatible water-miscible polar dispersant, and    -   b) combining the resulting dispersion with sufficient aqueous        solvent for the particles to convert them to a cohesive        hydrogel,        other embodiments of the invention include like methods:    -   wherein the water-miscible polar dispersant is water and the        aqueous solvent for the particles is acidified water;    -   wherein the water-miscible polar dispersant comprises ethanol;    -   wherein the water-miscible polar dispersant comprises        isopropanol or acetone;    -   wherein the polysaccharide particles have been crosslinked;    -   wherein the polysaccharide particles have been dehydrothermally        crosslinked;    -   wherein the polysaccharide particles have been crosslinked using        a separate crosslinking agent;    -   wherein the polysaccharide particles are uncrosslinked;    -   wherein the polysaccharide particles are substantially        collagen-free;    -   wherein the polysaccharide particles are substantially a single        polysaccharide;    -   wherein the polysaccharide particles are a blend of two or more        polysaccharides;    -   wherein the polysaccharide comprises cellulose, chitosan, agar,        alginate, carrageenan, chitin, chondroitin sulfate, dextran,        galactomannan, glycogen, hyaluronic acid, starch or mixture        thereof,    -   wherein the polysaccharide comprises an oxidized polysaccharide        or salt;    -   wherein the polysaccharide comprises carboxymethylcellulose;    -   wherein the polysaccharide is substantially only        carboxymethylcellulose;    -   wherein the polysaccharide comprises chitosan;    -   wherein such chitosan is unmodified;    -   wherein the polysaccharide comprises a mixture of chitosan and        another polysaccharide;    -   wherein the polysaccharide comprises a mixture of        carboxymethylceltulose and chitosan;    -   comprising forming the cohesive hydrogel without visible clumps        of unhydrated polysaccharide;    -   further comprising injecting or spraying a layer of the cohesive        hydrogel onto mucosal tissue;    -   further comprising injecting or spraying the cohesive hydrogel        into an opening, recess, passageway or joint in a limb; or    -   further comprising injecting or spraying the cohesive hydrogel        into an opening, recess, passageway or joint in a spinal column.

Although specific embodiments have been illustrated and described hereinfor purposes of description of the preferred embodiments, it will beappreciated by those of ordinary skill in the art that a wide variety ofalternate or equivalent implementations calculated to achieve the samepurposes may be substituted for the specific embodiments shown anddescribed without departing from the scope of the present invention.This application is intended to cover any adaptations or variations ofthe preferred embodiments discussed herein. Therefore, it is manifestlyintended that this invention be limited only by the claims and theequivalents thereof.

The invention claimed is:
 1. A composition comprising a dry,free-flowing, sterilized mixture of separate rehydratable particlescomprising about 20 to about 95 wt. % of chitosan particles containingamino groups and about 80 to about 5 wt. % of oxidized polysaccharideparticles containing aldehyde groups in sealed packaging, wherein theamino group functionality and molecular weight of the chitosan and thealdehyde group and molecular weight of the oxidized polysaccharide aresuch that the mixture of chitosan particles and separate oxidizedpolysaccharide particles are rehydratable in situ when removed from thesealed packaging and applied in a dry state to a surgical tissue site orwound moistened with bodily fluids such that the mixture of chitosanparticles and separate oxidized polysaccharide particles will adhere tosuch tissue site or wound and will adhere to one another byinter-particle crosslinking reaction of amino groups on the chitosanparticles with aldehyde groups on the polysaccharide particles, whereinthe dry, free-flowing, sterilized mixture of chitosan and oxidizedpolysaccharide particles contains less than 10% wt. % water.
 2. Acomposition according to claim 1 wherein the chitosan particles have anumber average molecular weight of about 10 to about 500 kDa.
 3. Acomposition according to claim 1 wherein the oxidized polysaccharideparticles comprise oxidized starch particles.
 4. A composition accordingto claim 1 wherein the mixture contains about 80 to about 20% of thechitosan particles and about 20 to about 80% of the oxidizedpolysaccharide particles.
 5. A composition according to claim 1 whereinthe mixture contains about 60 to about 40% of the chitosan particles andabout 40 to about 60% of the oxidized polysaccharide particles.
 6. Acomposition according to claim 1 wherein the mixture contains about 60to about 40% of the chitosan particles and about 40 to about 60% ofoxidized starch particles.
 7. A composition according to claim 1 whereinthe chitosan particles and oxidized polysaccharide particles arenon-comminuted.
 8. A composition according to claim 1 wherein thechitosan particles and-oxidized polysaccharide particles in the sealedpackaging are uncrosslinked.
 9. A composition according to claim 1wherein the chitosan particles and oxidized polysaccharide particles arerehydratable in situ to form a gel on the tissue site or wound.
 10. Acomposition according to claim 9 wherein the chitosan particles andoxidized polysaccharide particles provide a polysaccharide gel whenrehydrated.
 11. A composition according to claim 1 wherein the chitosanparticles and oxidized polysaccharide particles contain a sufficientlylow amount of collagen so as not to pose a potential risk oftransmission of or infection with bovine spongiform encephalopathy (BSE)or variant Creutzfeldt-Jakob disease (vCJD).
 12. A composition accordingto claim 1 wherein the dry, free-flowing, sterilized mixture of chitosanand oxidized polysaccharide particles are capable of forming asubstantially continuous conformal protective layer over such tissue.13. A composition according to claim 12 wherein the tissue comprisesmucosal tissue.
 14. A composition according to claim 13 wherein themucosal tissue is in a nasal cavity.
 15. A composition according toclaim 13 wherein the mucosal tissue is in a sinus cavity.
 16. Acomposition according to claim 12 wherein the tissue comprises aninjured, inflamed or surgically repaired surface and the mixture assistsin returning such injured, inflamed or surgically repaired surface to anormal state.
 17. A composition according to claim 16 wherein the returnof such surface to a normal state includes reciliation.
 18. Acomposition according to claim 1 further comprising a sustained releasetherapeutic agent.
 19. A composition according to claim 1 wherein themixture of particles is inherently antimicrobial without requiringaddition of a separate antimicrobial agent.